Paeoniflorin preparations and uses thereof for fat reduction

ABSTRACT

Disclosed are methods and preparations useful for reducing fat at a targeted area(s) on a human. The preparations comprise as an active ingredient an adipolysis enhancing (i.e., fat-melting) amount of an active ingredient, paeoniflorin (PF). The preparations may be provided as an injectable preparation or as a topically applied preparation, such as in the form of a crème or lotion. In topical preparations, the active ingredient paeoniflorin may be contained within nanospheres, such as albumin nanospheres. The PF-containing preparations may also include a permeant, such as azone. The method may be accompanied by the application of ultrasound to the area being treated prior to, during or after, or prior to, during, and after application of the paeoniflorin preparation to an area of the body in which fat reduction is desired. By way of example, the methods and preparations are effective for reducing targeted fat deposits at various anatomical sites of the body, such as the midsection (“love handles”), jowls, hips, arms, thighs and buttocks area.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of and claims priority to U.S. patentapplication Ser. No. 13/847,944 filed Jul. 10, 2013, and currentlypending, which is a divisional of U.S. patent application Ser. No.13/081,278, filed Apr. 6, 2011, now issued U.S. Pat. No. 8,491,947,which is a divisional of U.S. patent application Ser. No. 12/126,246,filed May 23, 2008, now issued U.S. Pat. No. 7,943,187. The entiredisclosures and contents of all the above applications are incorporatedherein by reference.

BACKGROUND

Supplements, medicines and other products that promote fat loss continueto be in great demand. Products suitable for reducing fat in targetareas including the thighs, stomach and midsection (“love-handles”), areof particular interest. However, existing fat loss products areincapable of accomplishing this result, and routinely require strenuousdiet and exercise to accomplish the desired results. Most fat lossproducts facilitate only a general fat loss across the body, rather thanin specific, targeted areas. A significant amount of time and effort istypically required in order to accomplish even modest, non-targetspecific weight loss.

An ever-growing percentage of the population continues to grow more andmore overweight. The absence of effective and safe over-the-counterweight-loss products has created an ever growing need for alternative,more effective methods for weight and/or fat control. Preferably, animproved weight loss product that satisfies these needs would closelymimic natural fat loss processes and target typical “fat-zone” proneareas. Such a product would typically also include a user-friendlydelivery device(s) to further enhance administration of the product to atargeted body area to reduce stored fat deposits under the skin.

These factors evidence a continuing and growing medical need for safeand effective non-prescription preparations for facilitating fat and/orweight loss, particularly targeted fat and/or weight loss.

SUMMARY

The present invention satisfies these and other needs in the art.

In one aspect, a method is provided for enhancing fat loss in an animalparticularly a human, by administering a preparation and/or compositioncomprising as an active ingredient paeoniflorin (PF), which is anatural, purified bioactive glucoside in Paeonial Radix (PR). PF may befurther described as comprising a natural, purified bioactive glucosideisolated from Paeonial Radix (PR), the roots of Pneonin Pall. In someaspects, the composition may be further described as comprising anadipolysis promoting amount of paeoniflorin (PF).

An adipolysis promoting amount of active ingredient comprisingpaeoniflorin (PF) may be described as an amount of the paeoniflorinformulation/preparation that evidences at least some reduction in themass or volume, of an adipose (fat) containing area when applied in oron that area. By way of example, an adipose containing area may bedescribed as an area of the body on which adipose (fat stores) isevident, such as the buttocks, stomach, hips, thighs, jowls, midsection,or any area in close proximity to these areas.

Triglycerides:

In a general and overall sense, the fat-burning, or adipolysis promotingactivity of the present preparations/compositions is provided by theactivity found to exist in particular preparations and/or formulationsof PF for inducing the expression of β-adrenergic receptors present onfat cells. This results, among other fat break-down promoting events, ina reduction in fat stores that closely resembles the physiological andbiochemical events that are known to occur during natural fat loss. Oneof the clinical indicators of the physiological events associated withfat breakdown is a decrease in detectable plasma levels oftriglycerides. The breakdown of triglycerides results in the breakdownproducts of glycerol and fatty acids. Thus, a clinical indication of fatbreakdown in a patient may be a reduction and/or lower plasma level oftriglycerides. Triglycerides in the blood plasma of a patient may thusbe monitored in a plasma sample from a patient as an indicator and/ormonitoring event of fat break down.

In some embodiments, an adipolysis promoting amount of PF may bedescribed as an amount of PF that is sufficient to provide an increasein the amount of glycerol released in a culture of 3T3-L1 adipocytes inthe presence of an adipolysis enhancing amount of PF, compared to theamount of glycerol released by 3T3 adipocytes in culture media withoutthe same amount of PF. By way of example, an adipolysis enhancing amountof PF may be an amount of about 1 umol/L to about 5 umol/L of the PFpreparation and/or composition.

In clinical studies, it is shown herein that a decrease in blood plasmatriglycerides is detectable upon treatment of the patient with PF, andis evidence of fat breakdown. In a particular embodiment, a reduction inplasma levels of triglycerides, and hence fat breakdown, results upontreatment of a patient with multiple, relatively small injections of PFwithin an area of a patient's fat stores with a total dose of about 0.5mg PF, each injection site receiving about 0.025 mg PF in a volume ofabout 0.5 ml injection grade water/Phosphatidyl choline.

cAMP Levels:

The natural fat break-down achieved with the presentcompositions/preparations may also be characterized by the increase incAMP levels that result as a consequence of treatment. Thus, in someembodiments, an adipolysis promoting amount of PF may be described as anamount of PF that is sufficient to increase levels of cAMP in a cultureof 3T3 adipocytes, compared to cAMP levels in a culture of 3T3adipocytes without the same amount of PF. By way of example, the amountof PF that will increase cAMP levels is about 11 moles/Liter.

Hormone Sensitive Lipase (HSL):

The natural fat break-down achieved with the presentcompositions/preparations may also be characterized by the increase inhormone sensitive lipase (HSL) levels that result as a consequence oftreatment with PF. Thus, in some embodiments, an adipolysis promotingamount of PF may be described as an amount of PF that increasesdetectable amounts of hormone sensitive lipase (HSL) in a culture of 3T3adipocytes, compared to hormone sensitive lipase (HSL) levels in aculture of 3T3 adipocytes without the same amount of PF. In someembodiments, this may be described as an amount of PF that increasesexpression of a gene encoding hormone sensitive lipase (HSL). By way ofexample, the amount of PF that increases detectable amounts of hormonesensitive lipase in a culture of 3T3 adipocytes compared to the amountof hormone sensitive lipase detectable in a culture of 3T3 adipocyteswithout PF is about 1 mmol/Liter.

Adipolysis-Promoting Ratio of Adrenergic Receptor (AR) Expression(lipolysis greater than lipogenesis), β-adrenergicreceptors/a2b-adrenergic receptor expression ratio.

The natural fat break-down achieved with the presentcompositions/preparations may also be characterized by the increase inexpression levels of β-adrenergic receptors that result as a consequenceof treatment. Therefore, in some embodiments, the adipolysis promotingamount of PF may be described as an amount of PF that is sufficient toprovide a lipolysis enhancing ratio of adrenergic receptor expression,relative to lipogenesis enhancing adrenergic receptor expression in aculture of 3T3 adipocytes without or absent the same amount of PF. Byway of example, the lipolysis enhancing adrenergic receptors includeAdrb1, Adrb2 and Adrb3, while the lipogenesis enhancing adrenergicreceptors include a2B-AR.

Generally, PF would be provided in an amount that would elicit alipolysis enhancing ratio of adrenergic receptor expression. By way ofexample, this amount would comprise an increase in the ratio ofβ-adrenergic receptor expression relative to a2b-adrenergic receptorexpression. This amount of PF may further be described as an amount thatresults in an increase in 3-fold to about 7 or 8-fold expression of thelipolysis enhancing adrenergic receptors, relative to the expressionlevel of a2b-adrenergic receptors.

Adiponectin Receptor (Adiporl):

The natural fat break-down achieved with the presentcompositions/preparations may also be characterized by a decrease inexpression levels of adiponectin receptor (Adiporl) that results as aconsequence of treatment. Therefore, and in some embodiments, theadipolysis promoting amount of PF may be described as an amount of PFthat is sufficient to provide a decrease or reduction in the expressionof Adipor 1 in a culture of 3T3 adipocytes relative to the amount ofAdiporl expression in a culture of 3T3 adipocytes without the sameamount of PF.

Obesity-Related Gene Panel:

Carboxypeptidase E gene (Cpe gene), peroxisome proliferator activatedreceptor gamma gene (Pparg, a regulator of adipocyte differentiation),adrenergic receptor genes (such as Adrb2, Adrb3) and Adiponectinreceptor 1 gene (Adiporl) are among some of the obesity-related genesthat comprise the obesity-related gene panel as described herein.

In these embodiments, an adipolysis promoting amount of PF may bedescribed as an amount of PF that increases the expression level of apanel of obesity-related genes relative to the expression level of thesame panel of obesity-related genes in the absence of the same amount ofPF. In some embodiments, the expression level of the obesity-relatedgene panel is increased about 1.5 fold, compared to the expression levelof this obesity-related gene panel in a culture of 3T3 adipocyteswithout this amount of PF.

By way of example, one of the genes that is an obesity-related gene asdescribed in the present methods and compositions is the gene encodingcarboxypetidase E (Cpe). In particular, an adipolysis promoting amountof PF may be described as an amount of PF that increases the expressionlevel of a Cpe gene by 3T3 adipocytes, compared to the expression levelof Cpe gene in a culture of 3T3 adipocytes without the same amount ofPF. By way of example, the amount of PF that increases the expressionlevel of Cpe gene by 3T3 adipocytes is an amount that will increaseexpression levels of Cpe gene 2 to 3 fold compared to the expressionlevel of Cpe gene in a culture of 3T3 adipocytes without this amount ofPF. In some embodiments, this amount of PF is about 1 umol/Liter.

Type of Delivery/Application Formulation:

In various aspects of the method, the preparation comprising the activeingredient PF may comprise PF in a concentration that is effective topenetrate the skin and into fat cells, such as to a subcutaneous (i.e.,just under the skin) deposit of fat cells. The PF will thus be providedin any variety of application modalities to provide an effective fatcell penetrating concentration of PF to a targeted fat area. This fatcell penetrating concentration of the active ingredient, PF, is furtherdescribed as an adipolysis promoting amount of PF. The adipolysispromoting amount of the PF as a component of a preparation and/orcomposition will be determined, at least in part, by the form of theparticular formulation (i.e., topical, injectable, etc.) as provided tothe targeted fat deposit area(s).

Where the preparation and/or composition is administered topically, suchas in a crème and/or lotion, the adipolysis promoting amount of the PFwill be a concentration comprising about 0.2 mg to about 0.3 mg per mlof the crème and/or lotion. The application of the crème and/or lotion,by way of example, may then be followed by the application of a weakelectrical current to the area.

In those embodiments where the preparation is in the form of a crèmeand/or lotion, the preparation may further comprise, for example, apermeant. Virtually any of a variety of permeants may be used inconjunction with the topical preparations of the invention. By way ofexample, such a permeant may comprise a permeant comprising azone. Apermeant is described as an ingredient that will assist in the abilityof the active ingredient PF to cross over and into the fat cells of thepatient fat area being treated.

Alternatively, the formulation as a crème and/or lotion may in someembodiments comprise albumin nanospheres. In this form, the preparationwould be applied to a desired area, followed by subjecting the area toultrasound. In this way, the albumin nanospheres in the compositioncrème and/or lotion will be driven through the skin to the fat cells bythe action of the ultrasound.

In those embodiments where the preparation is a preparation suitable forinjection, the preparation may be described as comprising aphysiologically compatible carrier solution, such as saline and/orinjectable grade water. In other embodiments, the injectable preparationwill further include phosphatidylcholine, or other similar phospholipidsor combination of phospholipids.

By way of further example, where the preparation and/or composition isto be administered as an injectable preparation, an adipolysis promotingamount of PF may be described as an amount that delivers about 0.02 mgof the PF per 0.5 ml. injection fluid.

In another aspect, a method of treating an animal to reduce fat areasand/or to increase and/or enhance adipolysis is provided. As part of anembodiment of the method, the injectable solution of the PF-containingcomposition and/or preparation is delivered subcutaneously and/orsubdermally to a target area of a patient at an injection site or sitesspaced at least about 1 cm or so between injection sites on the patienttarget area being treated. Where the preparation and/or formulation isto be injected, the method may first provide for the application of atopical anesthetic to the area, such as an anesthetic gel (lignocainointment or lidocain gel) for about 5 minutes prior to injection, tonumb an area that will receive one or more injections. The skin is firstwiped with an alcohol swap, 2 minutes or so later, a local anestheticointment such as gel anesthetic, is then rubbed on the surface area tobe injected. Five (5) minutes later, the contents of a 10 ml syringecontaining the PF injectable solution is then injected into about 20injection sites on the patient fat area being treated, with 0.5 ml. ofthe injectable PF solution being injected subcutaneously at eachinjection site.

It is further contemplated that the formulations and/or preparations maybe provided to a fat deposit area in any one or combination of thesetreatment forms suitable for achieving the desired results. In areaswith large fat deposits, such as the stomach area or midsection (“lovehandles”), the concentration of PF may need to be increased in theinjectable solution preparation. For example, it is contemplated that 1mg of PF per 5 ml of injectable water will be prepared.

In another aspect, the preparation and/or composition will comprise asan active ingredient a component PF having the following Formula I:

Wherein R is a monosaccharide. In some embodiments, the monosaccharideis glucose (Glc), galactose (Gal), or other monosaccharide. Inparticular embodiments, the monosaccharide is glucose (Glc). In someembodiments, the glucose may be described as the D-glucose stereoisomerof glucose.

The preparations and/or compositions may also further comprise otheringredients recognized by those of skill in the pharmaceutical arts forimproving delivery, stability of the formulation (shelf life),consistency, dispersion on the skin surface, scent, color, etc. By wayof reference, such formulation techniques and additives are described inRemington: The Science and Practice of Pharmacy, ALFONSO R. GENNARO,20th Edition. Baltimore, Md.: Lippincott Williams & Wilkins, 2000. Thisreference insofar as these teachings are concerned is herebyspecifically incorporated by reference.

The PF was purchased in a commercial purified dry form (powder) from acommercial vendor.

In other aspects, methods of reducing the size and/or dimensions oftargeted adipose (i.e., fat) stores and/or adipose tissue deposits areprovided. In some embodiments, this method comprises administering anadipolysis promoting amount of a preparation and/or compositioncomprising an adipolysis enhancing amount of an active ingredient, PF,to a targeted fat deposit area on a patient. The preparation may beadministered one or more than one time, such as in part of an injectionprotocol, until a desired reduction in a targeted fat deposit isobserved.

The injection protocol may comprise 10, 20, 30 or more than 30individual injections per session, and include 1, 2, 3 or more sessionsof such a regimen until the desired results are achieved. In someembodiments, a session of treatment for 20 injections. On average, it isdemonstrated that a noticeable reduction in measurable fat dimensionsmay be observed after 4 sessions of 20 injections each (20 injectionsconstituting one session) to 5 sessions of 20 injections. In someembodiments, about 0.025 mg PF is provided in a volume of about 0.5 mlof a carrier (such as sterile water, etc.) to each of 20 differentinjection sites on a patient as part of a single treatment session.

On average, a fat loss of about 3 cm may be achieved after 5 sessions ofthe injection series with the PF injectable preparations. In humans, thepresent preparations of PF provide for reducing or melting away about 1cm of fat in stomach and thigh areas per 1 to 1.5 sessions, and about 1cm of fat in the upper arms area per session. The methods also providefor a reduction in total cholesterol blood levels. In particular, asignificant reduction in serum cholesterol, and serum triglycerides wasobserved upon treatment with the herein described PF treatment methodsin humans. As part of the provided methods, a treated patient animalwould be encouraged and/or required to walk and/or exercise. By way ofexample, such may include a brisk walk of from 25 to 30 minutes per day,for a period of seven (7) or more days. In addition, the patient animalshould further drink two (2) or more liters of water for the same periodof time as the walking and/or exercise regime, to maximize the slimmingeffect of the treatment and to facilitate flushing of broken-down fatproducts out of the body. By way of example, it is envisioned that anarea on a patient that would be targeted as part of the method wouldinclude the thighs, hips, chin, jowls, stomach, midsection(“love-handles”), or any combination of these areas at the same time orsequentially in any order desired. In yet other aspects, a devicesuitable for the treatment of a patient animal to achieve fat loss isprovided. By way of example, such a device may comprise a syringe-likedevice having attached thereto a needle suitable for injection into atissue (See FIG. 11). LDL cholesterol, having attached thereto a needlesuitable for injection into a tissue (See FIG. 11).

The device in some embodiments will thus take the form of a syringe,such as a graduated barrel syringe cylinder capable of holding a volumeof a solution comprising PF, and a needle suitable for receiving one endof the graduated syringe cylinder. In particular embodiments, the needlewill be configured to be at a 90° angle relative to the syringecylinder. In some embodiments, the graduated barrel cylinder will have avolume capacity of 50 ml or more. In some embodiments, a 23× 11/4″0.65×32 mm needle may be used with a 25 ml “Slip Tip” non-lock syringefor administering the PF injectable preparations of the present methodsand kits.

The present invention, in yet another aspect, provides a kit thatcomprises the fat-melting, fat-reducing preparations as describedherein. In some embodiments, the kit may comprise 1 ml ampule or ampulesof PF and azone at a concentration of 0.2 mg/ml and a device suitablefor stimulating the entry of the PF in solution into fat cells of alocalized fat deposit on a patient. For example, such a device is theAlpha Wave healthtronic muscle stimulator. This small device istypically and routinely used by physiotherapist to stimulate musclesafter a sports injury by passing a low density current that causesmuscle contraction. Due to the low voltage current of about 2.5 amp, acurrent will be transmitted that will drive the PF through the skin andinto the adipose tissue after the azone disrupts an organized lipidlayer.

By way of example, and not limitation, the following are among some ofthe many advantages and features of the present preparations andmethods:

1. Acts directly on fat cells and dissolves fat in a manner that mirrorsthe physiologic fat loss.

2. Burns fat through activating receptors present on the fat cells inthe target area, responsible for triggering the intracellular cAMPpathway, PKC and HSL to dissolve fat naturally.

3. Increases the number of cellular receptors responsible for burningfat.

4. Breaks fat down into its natural byproducts.

5. Noninvasive, painless and area specific.

Many points of distinction exist between the present formulations andmethods for fat reduction and other methods described in the art. Forexample, while previous methods depend in part or whole on achievingsome level of appetite suppression or inhibition of fat absorption fromthe gut, the present formulations and methods do not.

In addition, the present methods/formulations for fat loss are distinctand superior in many ways to mesotherapy. Mesotherapy is the use ofintra- or subcutaneous injections containing mixtures of compounds totreat local medical and cosmetic conditions, and does not provide atreatment for any particular condition. It primarily involves a methodof drug delivery (Rotunda and Kolodney 2006), with the compounds used inmesotherapy comprising phosphatidylcholine (a compound derived from soy,and is a component of cell membranes in humans/other organism) mixedwith deoxycholate (bile salt), and may further be mixed withaminophyline. Through the use of punch biopsies taken at one and twoweek time points after treatment with mesotherapy, a loss insubcutaneous fat was reported. The subcutaneous fat loss was postulatedto have been accomplished by emulsifying fat deposits via aninflammatory necrosis and resorption (Rose and Morgan, 2005). Incontrast, and among other distinctions, the present methods provide forfat loss in a manner that mimics physiological fat break down, such asby break down into fat's natural byproducts, such as glycerol, viaincrease in a physiological second messenger molecule, cyclic AMP.

Additional features and advantages are described herein, and will beapparent from, the following Detailed Description and the figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 demonstrates PF increased the release of glycerol by 3T3-L1adipocytes significantly by the 7^(th) day after exposure.

FIG. 2 demonstrates intracellular cAMP tested by ELISA after incubationwith PF in 3T3-L1 adipocytes.

FIG. 3A-3B demonstrate expression of HSL in 3T3-L1 adipocytes afterincubation with PF. FIG. 3B, PF is shown to increase the expression ofHSL in 3T3-L1 from the 3^(rd) day of exposure, which follows theincrease of cyclic AMP and gives rise to release in glycerol as abyproduct of fat metabolism.

FIG. 4 demonstrates pathway of adipolysis.

FIG. 5 demonstrates Expression of AR in 3T3-L1 adipocytes exposed to PF.

FIG. 6A-6B demonstrate mechanisms of lipolysis stimulation by 13-ARs.6A=Basal State, 6B=Activated State.

FIG. 7 demonstrates an expression of Cpe in 3T3-L1 adipocytes exposed toPF.

FIG. 8 demonstrates an expression of Adiporl in 3T3-L1 adipocytesexposed to PF.

FIG. 9 demonstrates an expression of Pparg in 3T3-L1 adipocytes exposedto PF.

FIG. 10 demonstrates the clinical effect: Comparison of PF. Serumcholesterol, LDL, Cholesterol, serum triglycerides in blood of eightpatients was reduced after PF treatment.

FIG. 11 demonstrates a needle at 90° angle to the long axis of thesyringe.

FIG. 12 demonstrates glycerol release after incubation for 3 days withMesotheraphy (M) and PF.

FIG. 13 demonstrates glycerol release after incubation for 7 days withMeso (M) and PF. PF increased the release of glycerol significantly bythe 7^(th) day after stimulation (p<0.01).

FIG. 14 illustrates intracellular cAMP levels measured by ELISA afterincubation with Meso (M) and PF for 72 hours. In the PF group,intracellular cyclic AMP was significantly increased by PF in the firstday of stimulation (p<0.01). In the M group, the level of cAMP did notshow much difference compared to the control group.

FIG. 15 present results of ELISA demonstrating the intracellular cAMPafter incubation of fat cells (adipocytes) with Meso (M) and FF for 24hours. In the PF group, intracellular cyclic AMP was significantlyincreased by PF in the first day of stimulation (p<0.01). In the Mesogroup (M), the level of cAMP did not show much difference compared tothe control group.

FIG. 16 presents the expression of HSL in 3T3-L1 adipocytes afterincubation with Meso (M) and PF. PF increased the expression of HSL in3T3-L1 adipocytes from the third day after stimulation.

DETAILED DESCRIPTION

The present invention, in a general and overall sense, provides avariety of preparations and methods for enhancing lipolysis and fatreduction and/or loss in vivo in a patient animal. The preparationsand/or compositions include as an active ingredient PF. PF is a natural,purified bioactive glucoside in Paeoniac Radix (PR), the roots ofPaeonia Pall. PF, a natural plant extract purified to over 99%, isprovided here as a natural organic potent lipolysis drug. Its mode ofaction is through significantly enhancing the expression of severalobesity-related genes such as Adrb2, Adrb3, Cpe, Adiporl and Pparg.

PF enhances the expression of β-adrenergic receptors 2 & 3. β-adrenergicreceptor 2 is a major biolytic receptor in human fat cells.(β-adrenergic receptor 3 is important in regulating thermogenesis andlipolysis in brown adipose tissue through autonomic nervous system (ANS)activity. Its biologic intracellular pathway showed that activation ofβ-adrenergic receptors goes through the cAMP pathway which in turnsignificantly enhances the level of expression of Hormone-SensitiveLipase (HSL), to break down tri-glycerides into glycerol and fattyacids.

According to the present compositions/methods, PF significantly enhancesfat loss by enhancing a fat cell's ability to burn fat into its naturalby-products. PF can be administered in any of a variety of ways thataccomplishes contact of the preparation with fat cells or a tissuecomprising at least some fat cells. By way of example, and notlimitation, the composition may be provided to an animal, such as ahuman, by subcutaneous injections, or by applying the preparation as acrème and/or lotion. As a crème and/or lotion, the PF may be driventhrough the skin to the fat cells. This may be accomplished, forexample, by using a weak electric current similar to that used by aphysiotherapist, or by mixing PF with albumin nano spheres and drivingthe mixture through the skin to the fat cells using ultrasound.

To test the effectiveness of PF on targeted fat loss, a 3T3-L1 cell linewas used. The 3T3-L1 cell line provides an art-accepted model for fatloss. Fat loss is identified utilizing an in vitro adipolysis (digestionof fats) measure in a Swiss 3T3 mouse cell line. 3T3-L1 cells propagatedunder normal conditions have a fibroblastic phenotype. However, whentreated with a combination of dexamethasone, isobutylmethylxanthine(IBMX) and insulin, 3T3-L1 cells adopt a rounded phenotype andaccumulate lipids intracellularly in the form of lipid droplets.

As detailed below, PF was found to significantly increase the number ofβ-adrenergic receptors (β 1, 2 and 3 subtypes) responsible for the breakdown of fat into its natural byproducts. The exposure of 3T3-L1 fatcells to PF causes a significant increase in the level of expression ofintracellular cyclic adenosine monophosphate (cAMP) on days 1 & 3(P<0.01). This is followed by a significant increase in the level ofexpression of Hormone-Sensitive Lipase (HSL). HSL is a multifactorialtissue lipase that plays a critical role in fat metabolism. Such asignificant increase in HSL expression is followed by a significantincrease in glycerol release on day 7 of exposure to PF in vitro. Invivo, lowered levels of plasma triglycerides provide a clinicalindicator of fat breakdown in a patient.

The present preparations are demonstrated to possess clinicaleffectiveness in patients, and to provide effective site-specificlipolysis (fat breakdown) such as that stored in the midsection region(“love handles”), stomach, jowls, hips and thighs. Blood analysis and acomparison of before and after treatment with PF revealed that bloodsugar levels decreased upon treatment, and low-density-lipoprotein (LDL)levels showed a tendency towards reduction, compared to pretreatmentblood sugar level measures and LDL levels.

Accordingly, the present preparations, formulations, methods andtechniques provide among other things, the following advantages,characteristics and features:

1. A purified, natural and effective product (PF) that dissolves fatstored subcutaneously in the adipose tissue upon direct contact.

2. A predictable mode of PF's action. PF activates the expression of βadrenergic receptors as well as other fat metabolism genes.

3. A fat-reducing product that is provided in a suitable carrier todeliver PF across the skin to the fat layers.

4. A method that employs electric current and a charged carrier toeffectively deliver the preparation with the active agent, PF, to thefat cells and into the fat layers in target areas.

5. A method that delivers PF to fat cells through the use of albuminnanospheres driven through the skin by ultrasound.

In those embodiments where the preparation is a preparation suitable forinjection, the preparation may be described as comprising aphysiologically compatible carrier solution, such as saline and/orsterile water. In other embodiments, the injectable preparation willfurther include phosphatidylcholine, or any other of a variety ofsimilar phospholipids and combinations of phospholipids.

Example 1: PF Promotes Adipolysis

The present example demonstrates the utility of the present formulationsand/or preparations for promoting adipolysis, or fat break down.

The 3T3-L1 cell line is an accepted model by those of skill in the artfor natural fat loss. This cell line is a substrain of Swiss 3T3 mousecell line 3T3-L1. This cell line propagated under normal conditions hasa fibroblastic phenotype. However, when treated with a combination ofdexamethasone, isobutylmethylxanthine (a non-specific inhibitor ofphosphodiesterases) (IBMX) and insulin, 3T3 cells adopt a roundedphenotype and accumulate lipids intracellularly in the form of fatdroplets.

Differentiated 3T3-L1 adipocytes were cultured in media containing 1umol/L PF as test group, and media without PF as control. As a result oflipolysis activity, glycerol will be generated as a result oftriglyceride breakdown and released into the extracellular space.Glycerol content from 3T3-L1 adipocyte cell culture media may thereforeprovide an indicator of adipolysis. It was found that PF increased therelease of glycerol significantly by the 7^(th) day after exposure(p>0.01) (FIG. 1), compared to glycerol release from 3T3-L1 adipocytesin the absence of the same amount of PF.

It was found that PF increased adipolysis within one week afterstimulation was initiated. The PF preparations are thus demonstrated toinduce adipolysis indirectly by causing changes in gene expression thatlead to triglyceride breakdown. Further studies were carried out todefine the mechanism of action of PF.

Example 2: PF Increased cAMP

The present example, among other things, demonstrates the ability of theinvention to provide effective fat loss through PF action on cyclic AMPlevels. 3T3-L1 adipocytes were cultured in medium containing 11 mol/L PFas test group and medium only as control. Cells were lysed by adding 0.1N HCL, and intracellular cyclic AMP was measured by ELISA. It was foundthat intracellular cyclic AMP was significantly increased by PF in thefirst day of exposure (P<0.001), and the third day as well (P<0.01)(FIG. 2).

The major pathway leading to lipolysis involves activation ofcAMP-dependent Protein kinase A (PKA), which in turn activates othersubstrates such as HSL and perilipin. The agonists of β-adrenergicreceptor bind to the F3-adrenergic receptor, which activates theG-protein, Gs. The activation of Gs stimulates adenylate cyclase (AC) toproduce cyclic AMP. Protein kinase A (PKA) is activated by cAMP tophosphorylate the lipid droplet surface protein, perilipin (PL).Hormone-sensitive lipase (HSL) docks onto phosphorylated PL and breaksdown triglyceride into glycerol and free fatty acid. Glycerol isreleased into the extracellular space through aquaporin adipose (AQPad).The release of glycerol was found to be increased significantly by the7th day after PF stimulation (P<0.001), and that intracellular cyclicAMP was significantly increased by PF in the first day of stimulation(P<0.001), and the third day as well (P<0.01). The change andtime-relationship of cyclic AMP and glycerol release demonstrates thatPF induced adipolysis functions through the pathway of “secondmessenger” (cyclic AMP).

Example 3: Hormone Sensitive Lipase (HSL)

The present example demonstrates an increase in adipolysis in vivo asdemonstrated by an increase in detectable levels of hormone sensitivelipase (HSL).

3T3-L1 adipocytes were cultured in medium containing 1 mol/L PF as atest group and medium only as the control group. Total RNA was extractedand reverse transcribed to cDNA employing conventional methods known tothose of skill in the art.

Primers used for polymerase chain reaction (PCR) amplification of mouse

HSL primers were selected based on published sequence (NM_010719)(expected PCR fragment: 409 bp):

forward primer,  5′-GCTGGTGCAGAGAGACAC-3′; reverse primer, 5′-GAAAGCAGCGCGCACGCG-3′

For semi-quantitative analysis, the amplification cycles were chosenwithin the linear range (HSL: 24 cycles with denaturation at 58° C.,GAPDH: 21 cycles with denaturation at 58° C.). It was found that the PFincreased the expression of HSL in 3T3-L1 cells from the third day afterexposure (FIG. 3).

Hormone-sensitive lapse (HSL) is a multifunctional tissue lipase thatplays a critical role in the process of fat metabolism. The enzyme hasbroad specificity, catalyzing the hydrolysis of tri-, di-, andmonoacylglycerols, as well as cholesterol esters. HSL is thought tocatalyze the major rate-limiting step in lipolysis. The lipase isacutely activated by cAMP-dependent phosphorylation, which also leads toits redistribution from the cytoplasm to the lipid droplet. Regulationof adipocyte HSL is the primary means by which lipolytic agents, such ascatecholamines, stimulate the release of free fatty acids and thuscontrol circulating levels. In this study, PF was found to increase theexpression of HSL in 3T3-L1 from the third day of exposure, whichfollows the increase of cyclic AMP and gives rise to release of glycerolas a byproduct of fat metabolism (FIG. 4) in vitro.

Example 4: Nanosphere Formulation with PF

The present example is provided to demonstrate, by way of example only,one of the dermal preparations of PF. In particular, a nanosphereformulation with PF is presented.

Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC)for the topical application to the skin are made of lipids such asglycerol behenate (Compritol® 888 ATO), glycerol palmitostearate(Precirol® ATO 5), or the wax, cetyl palmitate. For NLC at roomtemperature, liquid lipids such as medium chain triglycerides (Miglylol®812) are added. Alternatively, oleic acid belonging to the frequentlyused penetration enhancers in semisolid vehicles applied to the skin,may be added to enhance drug uptake further (Lombard Borgia, Regehly etal. 2005). Mean particle size ranges of the nanospheres was from 50 to1000 nm. Nano dispersions contained 5 to 40% lipid. The higherconcentrated preparations are of semisolid appearance, and arecosmetically acceptable as they are. Depending on the mode andconcentration of the lipid, 0.5 to 5% surfactant may be added forphysical stabilization of the particles. For dermal use, these arePoloxamer 188, Polysorbate 80, lecitihin, Tyloxapol®, TegoCare® 450(polyglycerol methylglucose distearate), Miranaol®Ultra C32 (sodiumcocoamphoacetate) or saccharose fatty acid ester.

To facilitate dermal application, fluid dispersions which are obtainedwhen the lipid content is low (<10%) can be incorporated into a crème orgel base which does not induce dissolution or aggregation of SLNparticles. Photon correlation spectroscopy and differential scanningcalorimetry results have not changed over a storage period of 6 months(Jenning, Thunemann et al. (2000) and Wissing and Muller (2001), copiedfrom Scafer-Korting, Mehnert et al. (2007)).

Example 5: Microarray Analysis of 111 Obesity-Related Genes

The present example demonstrates that one of the clinical indicators ofthe fat-burning and obesity fighting activity of the presentformulations and/or preparations includes an increase in the expressionlevels of obesity-related genes.

In order to explore a more detailed mode of action of PF on adipolysis,microarray analysis was carried out on a panel of obesity-related genes.Expression levels from this gene panel are shown to be increased by PF,compared to expression levels of these genes in the absence of PF.

3T3-L1 adipocytes were cultured in medium containing 1 umol/L PF as testgroup and medium only as control. Total RNA was extracted and OligoGEArray was tested using Superarray OMM-17. The GEArray includes 111obesity-related genes that are directly involved in the regulation ofenergy intake and expenditure. The genes included orexigenic peptides,hormones, and receptors, anorectic peptides, hormones, receptors, andcentral and peripheral signaling molecules involved in energyexpenditure. The increase changes or decrease changes of more than 1.5fold are regarded meaningful according to the diagnosis discipline, asdescribed in the insert literature of a metabolic disease/obesity genepanel product purchased from Superarray Bioscience Corporation.(Frederick, Md.).

In the following microarray analysis, the red color shows the expressionof obesity-related genes increased by more than 1.5 fold, and the bluecolor shows the expression of the obesity-related genes decreased bymore than 1 5 fold, i.e., the number less than 0 66 shows reduction ofmore than 1.5 fold. In the graphs, the data above the upper line and thedata below the lower line show a significant change of gene expression.

Adrenergic Receptor (AR)

The adrenergic system plays a major role in the regulation of lipolysisin white adipose tissue, which is the major site of energy storage.Catecholamines are able to stimulate lipolysis by the activation ofadipocyte 8-adrenergic receptors (131-, B2-, B3-AR). At the same time,catecholamines can also increase lipid storage through alb-AR. Since Band a2b-AR coexist on the same fat cell, the ratio of functional alb-and B-AR present in adipose tissue may deteimine whether fat storage orrelease is activated by catecholamines (Soloveva et al., 1997). Thepresent data of microarray analysis after exposure to PF demonstrated asignificant increase in the expression of Adrb1, Adrb2 and Adrb3, and noeffect on Adra2b (Table 1, FIG. 5). Adrb1 was activated earlier followedby Adbr3 then Adbr2. Adrb2 and Adrb3 were increased by 7.4 fold and 5.65fold, respectively, at 7 days post exposure to PF. Since the expressionof Adra2b almost did not change during the whole study, while there wasa significant increase in 13 receptor levels, that the ratio of13-adrenergic receptors to a2b-adrenergic receptors is shown to beincreased by PF. In other words, lipolysis exceeds lipogenesis whenexposed to PF.

Table 1. Ratio of expression signals of AR in 3T3-L1 adipocytes exposedto PF compared to control

TABLE 1 Expression of AR in 3T3-L1 adipocytes exposed to PF Control PF-3days PF-6 days PF-7 days β1-adrenergic 1 3.39 0.8 0.58 receptor (Adrbl)β1-adrenergic 1 0.74 1.96 7.40 receptor (Adrb2) β1-adrenergic 1 1.563.18 5.65 receptor (Adrb3) β1-adrenergic 1 0.92 1.44 1.27 receptor(Adra2b)

The three β-adrenergic receptor subtypes β1-, β2-, β-AR) are members ofa large family of G protein-coupled receptors, which function throughthe production of cAMP and the activation of HSL (Soloveva et al.,1997). In the basal state (FIG. 6A), nonphosphorylated HSL is in thecytosol probably bound to some cytosolic acceptors such as lipotransin,and nonphosphorylated perilipin (PL) is tightly bound to the lipiddroplet. HSLs do not have free access to the droplet. Whencatecholamines interact with the β-ARs (FIG. 6B), β-ARs alternativelycouple to G-protein, Gs. The activation of Gs stimulates adenylatecyclase (AC) to produce cyclic AMP. Protein kinase A (PKA) is activatedby cAMP to phosphorylate the lipid droplet surface protein, perilipin(PL). Hormone-sensitive lipase (HSL) docks onto phosphorylated PL andbreaks down triglyceride into glycerol and free fatty acid. Glycerol isreleased into the extracellular space through aquaporin adipose (AQPad)(FIG. 4).

The results of β-ARs gene expression and that of the change of cAMP andHSL (shown in part IV) mirror the pathway above.

Carboxypeptidase E (Cpe)

Carboxypeptidase E (Cpe) is a key enzyme involved in the biosynthesis ofpeptide hormones and neurotransmitters, including insulin. Cpe plays avital role in fat metabolism. The mutations in the gene of Cpe result in“fat mutation” (Naggert et al., 1995). “Fat mutation” represents thefirst demonstration of an obesity-diabetes syndrome elicited by agenetic defect in a prohormone processing pathway (Naggert et al.,1995). The fat mutation mouse does not express Cpe and presents as obeseand hyperglycemic. The present data of microarray analysis for PFdemonstrated a significant increase in the level of expression of Cpe onday 7 of exposure of 3T3-L1 adipocytes to PF. The expression of Cpe wasincreased by 2.67 fold compared to the control (Table 2, FIG. 7).

TABLE 2 Ratio of expression signals of Cpe in 3T3-L21 adipocytes exposedto PF compared to control Control PF-3 days PF-6 days PF-7 daysCarboxypeptidase E (Cpe) 1 0.79 1.04 2.67

Adiponectin has been shown to increase insulin sensitivity and decreaseplasma glucose by increasing tissue fat oxidation. AdipoR1 serves asreceptor for globular adiponectin and mediates increased AMP-activatedprotein kinase, glucose uptake and fatty-acid oxidation by adiponectin(Yamauchi et al, 2003). The present data of microarray analysis revealedthat the expression of Adiporl was increased by 1.86 fold on day 7 ofexposure of 3T3-L 1 adipocytes to PF (Table 3, FIG. 8). Since it isproved that obesity-linked down-regulation of adiponectin is a mechanismwhereby obesity could cause insulin resistance and diabetes, adiponectinreceptor agonists and adiponectin sensitizers are suggested to serve asversatile treatment strategies for obesity-linked diseases such asdiabetes and metabolic syndrome (Kadowaki and Yamauchi, 2005).

TABLE 3 Ratio of expression signals of Adiporl in 3T3-L1 adipocytesexposed to PF compared to control Control PF-3 days PF-6 days PF-7 daysAdiponectin receptor 1 1 0.68 1.21 1.86 (Adiporl)

Peroxisome Proliferators Activated Receptor Gamma (Pparg)

Pparg encoded PPAR-gamma, is a regulator of adipocyte differentiation.PPAR-gamma has been implicated in the pathology of numerous diseasesincluding obesity, diabetes, atherosclerosis and cancer. In other words,its reduction will inhibit the differentiation of new adipocytes. It wasshown by microarray that the level of expression of Pparg was reduced by1.54 fold on day 7 of exposure of 3T3-L1 adipocytes to PF (Table 4, FIG.9). In the microarray analysis, the blue color shows gene expressiondecreased by more than 1 5 fold, i.e. the number less than 0.66 showsreduction of more than 1.5 fold. In the graph, the data below the lowerline shows significant reduction of the gene expression.

TABLE 4 Ratio of expression signals or Pparg in 3T3-L I adipocytesexposed to PF compared to control Control PF-3 days PF-6 days PF-7 daysPeroxisome proliferators 1 0.91 1.09 0.64 activated receptor gamma(Pparg)

Example 6 Comparison of the Effect of PF and Meso (M) on Fat Cells

The present example demonstrates that the effect of PF and mesotheraphy(M) on fat cells is distinct from one another. The present exampleparticularly demonstrates that the mode of action of PF on lipolysis isdistinct from the action of mesotheraphy. PF breaks down fat byactivating fat cells to express more β-adrenergic receptors responsiblefor fat metabolism. Breaking down fat by PF is accompanied by anincrease in cAMP. The increase in cAMP in turn increases the level ofexpression of HSL, which breaks down fat and causes a significantincrease in glycerol released as a result of breaking down triglyceridesinto glycerol and fatty acids (See FIG. 12—Glycerol release afterincubation for 3 days with Meso (M) and PF). Mesotheraphy, on the otherhand showed no effect on most of the measured parameters, except onslightly enhancing the HSL as a result of the presence of aminophyline,which is known to act on β-adrenergic receptors.

There is evidenced a significant release of hormone sensitive lipase(HSL) when fat cells are exposed to PF. There is not a significantrelease of HSL upon Mesotherapy. The present application includes datain the attached figures that establishes that the use of PF results in arelease of glycerol from fat cells, that a resulting increase in cAMPoccurs and that an increase in hormone sensitive lipase (HSL) occurs.These events did not occur with mesotheraphy, and/or did not occur tothe extent evidenced with PF, in comparison to control cultures (no drugadded) or with mesotheraphy.

There is evidenced a significant increase in the expression of Adrb1,Adrb2 and Adrb3, and no influence on Adra2b, with exposure to PF. Adrb 1was activated earlier followed by Adbr3, then Adbr2. Adrb2 and Adrb3were increased by 7.4 fold and 5.65 fold, respectively, at 7 days postexposure to PF. Since the expression of Adra2b almost did not change,while there was significant increase in the 13 receptor levels, theratio of 13-adrenergic receptors to a2b

adrenergic receptors is increased by PF. Lipolysis therefore exceedslipogenesis upon exposure to PF. The data presented herein compares theeffects of PF as contrasted to the effects of mesotheraphy and controltreatments on cultures of 3T3 cells. This data demonstrates thatactivation of the intracellular pathway can only be triggered or inducedby the activation of the beta adrenergic receptors, and that activationof the beta adrenergic receptors was only achieved with PF exposure, andthere was no activation of beta adrenergic receptors with othertreatments.

Example 7 In Vivo Clinical Data

The present example demonstrates that the present formulations of PF aspart of an injectable preparation effectively provide for targeted fatreduction in a human.

Eight patients were treated with PF. The levels of three indices such asserum cholesterol, LDL Cholesterol, serum triglycerides in blood weretested before and after treatment. Serum cholesterol, LDL Cholesterol,serum triglycerides in blood for the eight patients were reduced afterPF treatment (Table 5, FIG. 10). For serum cholesterol, the averagelevel for these cases was higher than the high limit of normal range,and the average level fell into normal range after treatment, whichshowed significant change statistically (P<0.01). For LDL Cholesterol,serum triglycerides, although the change of pre-treatment andpost-treatment does not show statistical change, the extent of decreaseis meaningful at clinical aspect.

In human, PF has been found to dissolve 1 cm of fat per 1-1.5 sessionsin the stomach and thigh areas while about 1 cm per session was observedin the upper arms areas. In almost all the patients, the totalcholesterol blood levels were reduced significantly with the PFtreatment.

The average level of cholesterol in these cases was higher than the highlimit of normal range, and the average level fell into normal rangeafter treatment, which showed statistically significant change (P<0.01).

TABLE 5 Clinical effect comparison of PF Serum Serum Serum Serum SerumSerum Cholesterol Cholesterol Cholesterol Cholesterol CholesterolCholesterol (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) Case Pre-txNR: Post-tx Pre-tx NR: Post-tx Pre-tx NR: Post-tx No. 50-200 NR: 50-20060-150 NR: 60-150 35-160 NR: 35-160 1 216 171 123 113 62 73 2 173 135109 75 88 60 3 207 151 142 92 72 59 4 233 222 93 146 116 67 5 169 168 72108 54 71 6 269 237 208.5 176.5 83 110 7 180 150 117 98 70 55 8 176 148118 93 90 85 Ave 202.88 172.75 122.81 112.69 79.38 72.50 SD 35.37 37.0540.41 33.07 19.38 17.89 P 0.002 0.452 0.462

Example 8 In Vivo Fat Reduction

Examples of actual treatment results are presented below and the periodsof treatment. These patients were selected because they represented theresults of treatment of different areas in the body (stomach, arms, andthighs).

-   -   1) Patient 1: Period of Treatment=6 weeks/11 sessions    -   Measurements at the stomach area: Size before start: 90 cm    -   Size at End of Treatment: 83 cm    -   2) Patient 2: Period of Treatment=5 weeks/9 sessions    -   Measurements (at the stomach    -   area) Size before Start: 96 cm    -   Size at End of Tx: 90 cm    -   3) Patient 3: Period of Treatment=1 month/8 sessions In Both        Arms: 8 Sessions in total, (i.e. 4 sessions per side)    -   Areas treated: both upper arms    -   Size Before Start: 40 cm    -   Size at End: 36.5 cm

Complete Protocol for the Procedure Injectable Preparations Preparationof Solution

1 ml of PF (1) at a concentration of 1 mol is mixed with 4 ml injectablewater and 5 ml (phosphatidyl choline (PPC)) prepared from soya beanextract and 5 ml 2% lidocain. A total of 15 ml solution. This solutionis mixed together in a 20 ml syringe and the needle is changed to asmall insulin needle Gauge 30×½ as shown therein before the actualinjections are given.

Mode of Action of the Mixture

PF is the active ingredient in the mixture. It acts upon contact withthe fat cells in the adipose tissues. Phosphatidyl choline is a milddetergent that will bind to fat and thereby bring PF in contact with fatcells for an extended period of time (between 8-12 hours). The lidocainin the mixture increases vascularization into the injected site thusbring in more blood vessels to clear the dissolved fat byproducts.

A topical anesthetic is usually applied to the site prior to injectionsby 5-10 minutes.

The solution is then given subcutaneously directly into the fat areas ata dose of 0.5 ml per site totaling 20 injections. These injections arespread out to cover a big surface area, e.g. could be spread out througha whole midsection (“love handle”) in case of a larger patient, or ifthe patient were an average size it could be spread out to cover rightand left love handles in one session. For reference, please refer to thepatients injected in the thigh and buttocks area, for explanation. Thispatient was injected with 30 smaller injections of 0.2 ml per site, andthey were spread throughout the whole outer and inner thigh and buttocksareas on the right side.

Each of these 20 smaller injections comprises a session. On average, anoticeable difference was seen within 4-5 sessions. The initialcalculations measured 3 cm of fat loss in 5 sessions. The patient lostover 5 pounds of fat with 10 sessions.

Number of Sessions Required by the Patients

Each patient is different, depending on their size and the desiredamount of fat to be lost. Each 10 sessions resulted in about 7 cm of fatloss. Active patients and those who followed instructions showed aboveaverage fat loss. Some patients needed to sculpture small areas andtherefore discontinued treatment when the desired result was achieved.The addition of 2 sessions, together with an aerobic instructor, wasalso observed to result in a weight loss of over 3 pounds in 2 weeks.

Frequency of Injections

One patient evidenced more than 5 inches of fat loss, received 10sessions over about 6 weeks. After the desired results were achieved,the patient can receive one session every one to two months formaintenance.

Alternative Mode of Application

With morbidly obese patients, such as the patient results reportedabove, the dose of the active ingredient was doubled to 2 ml of 1 mmolin 3 ml of injectable water and mixed with the rest of the solution asmentioned above. A booster injection was used where smaller injectionswere not appropriate, and the content of one syringe was concentratedinto one or two areas of the fat and large needles were used in aretrograde manner of injection. For example, as the needle is pulledout, the injectable solution is released. The needle is rotated in acircular manner to cover a large surface area. It is important to notethat the needle is bent to a 90° angle to the long axis of the syringeas shown in FIG. 11.

About 3 ml is injected in this manner, and then the needle is withdrawn,but not totally out of the initial injection site, and re-inserted intoanother plane at about 30° in a counter clock-wise manner. This processis repeated about 5 times until the content is all injected. Forexample, employing a circle injection site area to be treated as atargeted fat area, with its center as the initial site of insertion ofthe needle, then the needle is rotated around this circle in the mannerdescribed above.

Concentration ranges of PF included in injectable preparations; doses ofPF provided to a patient at each treatment (injection) episode; specificsteps that were used in preparing the injectable preparations.

0.5 mg of PF was dissolved in 5 ml of injectable water rendering aconcentration of 0.1 mg/ml. This was then mixed with 5 ml ofPhosphatidyl Choline in a 20 ml syringe. The large gauge needle was thenreplaced with a smaller gauge (Gauge 27-30, in some embodiments, a 30gauge×½) insulin needle and the 10 ml was injected into 20 differentsites of a given area. 0.5 ml of the solution was injected into thestomach, with about 1 cm separating each injection site.

Concentration of PF included into the topical preparation: 0.2 mg to 0.3mg of PF were dissolved in 1 ml of azone liquid.

This mixture was then massaged into the target area. Azone has beenextensively studied using a range of permeates. The composition of theazone is C₁₈11₃₅NO. It is colorless, slightly yellowish or transparentin color, and is oil based. Heavy metal content in this compound is lessthan 0.001%. The azone used in the present studies was purchased fromNan Jing Long Tan chemical company, China. Azone has been shown toenhance permeability through the skin by disrupting the organized lipidstructure in the intercellular region of the stratum corneum. Thisprocess leads to increased lipid fluidity and enhanced drug diffusion.This is the reason azone was chosen to be mixed with PF.

A low current of 2.5 amp is then passed through pads placed on the skinof the desired treatment area which then drives the PF through the skininto the adipose tissues, thus allowing the PF to come in contact withthe fat cells.

Protocol Used in Creating Albumin Nanospheres

Many different preparations of nanospheres will be used in thepreparations of the present crèmes and topical preparations. Any numberof different nanosphere formulations known to those of skill in the artmay be used in the practice of the present invention. By way of example,such include any known delivery in the area of nanospheres.

Patient Preparation

For Regular Injections: After sterilization of the surface area with 74%ethanol, topical application of lignocain ointment or lidocain gel wasapplied for 5 minutes to numb the skin where the injections are to takeplace.

For Booster Injection: The patient is prepared using 1 ml of 2% lidocainto anaesthetize the site of entry of the large needle. This way, theprocedure is almost painless. 3-5 minutes later, the booster injectionis given slowly over a 3 minute period.

The patient should ingest a meal before the session, or is provided achocolate bar or a cookie before a session.

Patients are forewarned that bruising may occur due to the injections.Thus, some bruising in a patient may be expected. For example, bruisingwas observed in a patient injected in the thighs. The bruising, usuallydisappears within 1 week. In addition, redness and tenderness at theinjection site of the booster, is very common. Redness lasts for acouple of hours and the tenderness lasts for a couple of days. Bruising,is encountered more with the booster than the regular injections, butalso disappears within a week or so. Another observation is fatigue as aresult of treatment in some patients. Two patients suffered fromfatigue, with the booster session, but not with the regular session. Inaddition, a very slight rise in temperature, to about 37.1 or 37.2 for afew hours, was noted. Dizziness for a few minutes was also observed in afew patients, and the adjustable patient chair was reclined and theprocedure was continued uneventfully. No diarrhea was observed, butsofter stool was reported.

Protocol after the Sessions

Patients are asked to drink 2 liters of water per day throughout theday.

Patients were asked to walk or exercise for 25 minutes/day for the weekafter each session. This is intended to flush out dissolved fatbyproducts.

Avoid fatty diet, wine, alcohol, for the first 48 hrs after theinjections, to allow the blood to carry more of the fat byproducts to beexcreted.

A Second Mode of Application—Creme Preparations

The present preparations may be provided in the form of a crèmecontaining 1 ml of PF at a concentration of 1 mmol mixed with 1 ml ofazone which is then massaged into the target area. Azone has beenextensively studied using a range of permeants. Azone has been shown toenhance permeability through the skin by disrupting the organized lipidstructure in the intercellular region of the stratum corneum. Thisprocess leads to increased lipid fluidity and enhanced drug diffusion.This is the reason PF was mixed with azone. Furthermore, a low currentis then passed through the tissue area, which then drives the PF throughthe skin into the adipose tissues. PF may in this way come in contactwith fat cells. The clinical results are show below and the details ofeach patient is documented.

TABLE 6 AMOUNT OF NUMBER OF REDUCTION PATIENT AGE SEX TIMES IN CM #1 37M 6 3.5 cm above and below the umbilicus #2 40 F 16 9 cm above theumbilicus. 9 cm from midsection. 8 cm from waist #3 19 F 6 2 cm fromeach side of the upper thigh #4 32 F 7 8 cm from the right buttock & 9cm from the left buttock #5 25 F 8 9 & 10 cm from each leg below theknees #6 44 F 12 7 cm above umbilicus and 10 cm from midsection #7 19 F9 6 cm from left thigh and 8 cm from right thigh #8 24 F 30 1.5 size inbra size #9 44 F 20 6 cm above umbilicus and 9 cm below umbilicus #10 44F 20 5 cm from the waist and 5 cm from midsection #11 38 F 16 10 cmabove the umbilicus, 9.5 cm below the umbilicus and 5 cm from mid-section (love- handles) #12 30 F 8 10 cm from love handles and 6 cmbelow the umbilicus

Alternative Injection Protocol

The present example illustrates an alternative series of treatments thatwill comprise a treatment session for targeted fat reduction using theinjectable preparation and/or composition of PF. In this example, aseries of 20 small (0.1 to 0.2 ml) injections comprises a session oftreatment. On average, a noticeable difference can be seen in a patient,as evidenced by a reduction in size, within 4 to 5 sessions. Themeasurable fat loss was 3 cm of fat loss in 5 sessions.

In eight patients treated with PF as described here, the levels of 3indices such as serum cholesterol, LDL cholesterol, serum triglyceridesin the blood were examined before and after treatment. All of thesecholesterol parameters were reduced in these eight patient.

In a prior study with Mesotherapy, these cholesterol levels in bloodwere not reduced (Hexsel, Serra et al 2003).

For serum cholesterol, the average level for these cases was higher thanthe high limit of a normal range, and the average fell into normal rangeafter treatment, which showed a statistically significant change.(p<0.01). For LDL cholesterol, serum triglycerides, although the changeof pre-treatment and post-treatment did not show a statisticallysignificant change, the extent of decrease was clinically meaningful inthe management of the patient.

Example 9 Delivery Devices

The present example described particular devices and apparatus that maybe used in the delivery and application of the various PF topicalpreparations and injectable preparations of the present invention.

Sauna belt: Uzap tummy, butt, thighs (osim)

Input 100-240 V about 56 Hz

1.5 A 200VA

Output+24V−2.5 A

It has a remote with the following details:

Power consumption 60W

Operating voltage 24V d.c. 2.5 A

Alpha wave healthtronic muscle stimulator and exercise (the device thattransmits electrical current to the pads and is used after/beforeapplication of the PF containing crème)

Model BM-303

Power supply DC6V (batteries UM-1×4)

Watts 0.6

Injection Device: Syringe Barrel with 90° angle needle.

Specific steps that were used in preparing the injectable preparations:0.5 mg of PF was dissolved in 5 ml of injectable water rendering aconcentration of 0.1 mg/ml. This was then mixed with 5 ml ofPhosphatidyl Choline in a 20 ml syringe. The large needle was thenreplaced with a smaller gauge insulin needle and the 10 ml was injectedinto 20 different sites of a given area. 0.5 ml of the solution wasinjected into the stomach with about 1 cm separating each injectionsite.

Concentration of PF included into the topical preparation: 0.2 mg to 0.3mg of PF were dissolved in 1 ml of “Azone liquid”. This mixture was thenmassaged into the target area. Azone has been extensively studied usinga range of permeates. The chemical formula for azone is C₁₈H₃₅NO. It iscolorless, slightly yellowish, or transparent, and is oil based. Heavymetal content in this compound is less than 0.001%. Azone for thepresent study was purchased from Nan Jing Long Tan chemical company,China. Azone has been shown to enhance permeability through the skin bydisrupting the organized lipid structure in the intercellular region ofthe stratum corneum. This process leads to increased lipid fluidity andenhanced drug diffusivity. This is the reason PF was chosen to be mixedwith azone. A low current of 2.5 Amp is then passed through pads placedon the skin of the desired treatment area, which then drives the PF ofthe crème/lotion through the skin into the adipose tissues. In thismanner, PF is allowed to come in contact with the fat cells.

Example 10 Nanosphere Formulations

Many different preparations of nanospheres may be used in the practiceof the present preparations and compositions. The most effectiveformulations will then be identified.

Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC)for the topical application to the skin are made of lipids such asglycerol behenate (Compritol 888 ATO), glycerol palmitostearate(Percirol ATO 5), or the wax, acetyl palmitate. For NLC, at roomtemperature liquid lipids such as medium chain triglycerides (Miglyol812) are added. Alternatively, oleic acid belonging to the frequentlyused penetration enhancers in semisolid vehicles applied to the skin mayenhance drug uptake further. Mean particle size ranges from 50 to 1000nm. Nanodipsersions contain 5 to 40% lipid. The higher concentratedpreparations are of semisolid appearance and are cosmetically acceptableas they are. Depending on the mode and concentration of the lipid, 0.5to 5% surfactant have to be added for physical stabilization of theparticles. For dermal use, the preparations may also include, by way ofexample, the following: Poloxamer 188; Polysorbate 80; Lecithin;Tyloxapol; TegoCare 450 (polyglycerol methylglucose distearate); MiranolUlta C32 (sodium cocoamphoacetater); or saccharose fatty acid ester.

To facilitate dermal application, fluid dispersions which are obtainedwhen the lipid content is low (<10%) can be incorporated into a crème orgel base which does not include dissolution or aggregation of SLNparticles. Photon correlation spectroscopy and differential scanningcalorimetry results have not changed over a storage period of 6 months.

Example 11 Fat Reduction Kits

Component Pieces that would be a part of a kit according to the presentexample include:

-   -   1. 1 ml ampule of PF and Azone at a concentration of 0.2 mg/ml    -   2. device like the Alpha Wave healthtronic muscle stimulator &        exercise. This a small device used by physiotherapist to        stimulate muscles after a sports injury by passing a low density        current that causes muscle contraction. Due to the low voltage        current of about 2.5 Amp, a current will be transmitted that        will drive the PF through the skin and into the adipose tissue        after the azone disrupted the organized lipid layer.        It should be understood that various changes and modifications        to the presently preferred embodiments described herein will be        apparent to those skilled in the art. Such changes and        modifications can be made without departing from the spirit and        scope of the present subject matter and without diminishing its        intended advantages. It is therefore intended that such changes        and modifications be covered by the appended claims.

BIBLIOGRAPHY

The following references are specifically incorporated herein byreference.

-   Asaadi, M., A. P. Salas, et al. (2004). Mesoplasty: a new approach    to non-surgical liposculpture. American Society of Plastic Surgery,    Philadelphia, Pa., Oct. 9 to 13, 2004.-   Ablon G., Preliminary experience with mesotherapy utilizing    phosphatidylcholine. American Society for Dermatologic    Surgery-American College of Mohsmicrographic surgery and cutaneous    oncology combined annual meeting, 2005.-   Rittes, P. G. (2001). Dermatologic Surgery 27(4): 391-392-   Hexsel, D., M. Serra, et al. (2003). J Drugs Dermatol 2(5): 511-8.-   Guedes Rittes, P. (2003). Aesthetic Plastic Surgery 27(4): 315-318.-   Rotunda, A. M. (2004). Journal of the American Academy of    Dermatology 50(3S): 160-160.-   Rotunda, A. M., H. Suzuki, et al. (2004). Dermatologic Surgery    30(7): 1001-1008.-   Moy L S. Phosphatidylcholine injections. A study measuring decreased    subcutaneous fat thickness. American Society for Dermatologic    Surgery and the American Society of Mohsmocrographic surgery and    cutaneous oncology combined annual meeting, San Diego, Calif., Sep.    30 to Oct. 3, 2004.-   Rullan P, Hexsel D. Phosphatidylcholine injections for lipolysis of    neck and jowls: 50 case presentation. American Society for    Dermatologic Surgery-American College of Mohsmicrographic surgery    and cutaneous oncology combined annual meeting, Oct. 27 to 31, 2005.-   Duncan, D. I. and F. Hasengschwandtner (2005). Aesthetic Surgery    Journal 25(5): 530-543.-   EP1748780/W02005112942-   EP1021191/W09917712-   Duncan, et al (2005) Aesthetic Surgery, 25(5):530-543.-   Hexsel, et al (2005) Otolaryngologic Clinics of North America,    38(5):119-29.-   Jones, et al (1999) International Journal of Pharmaceutics 177(2):    137-159.-   Le Maire, et al (2000) BBA-Biomembrances 1508 (1-2): 86-111.-   Navder (1997) Life Sciences 61(19): 1907-1914.-   Rittes (2001) Dermatologic Surgery 27(4): 391-392.-   Rose, et al (2005) Journal of Cosmetic and Laser Therapy,    7(1):17-19.-   Salti, et al (2007) Dermatologic Surgery, 34(1):60-66.-   Vedamurthy (2007) Indian J Dermatol Venereol Leprol 73(1): 60-2.-   Jenning, et al (2000) International Journal of Pharmaceutics 199(2):    167-177.-   Lombardi, et al (2005) Journal of Controlled Release 110(1):    151-163.-   Schafer-Korting, et al (2007) Advanced Drug Delivery Reviews 59(6):    427-443.-   Wissing, et al (2001) Pharmazie 56(10): 783-6.

What is claimed is:
 1. A method for enhancing fat loss in a humancomprising: administering an adipolysis enhancing paeoniflorinpreparation comprising paeoniflorinin and a liquid carrier to a human;enhancing fat loss in the human, wherein fat loss in the human providedthe paeoniflorin preparation is enhanced compared to fat loss in a humannot provided the paeoniflorin preparation.
 2. The method of claim 1wherein the paeoniflorin preparation is not an injectable preparation.3. The method of claim 1 wherein the paeoniflorin preparation comprisesa concentration of paeoniflorin of about 0.02 mg/0.5 ml to about 0.25mg/0.5 ml.
 4. The method of claim 1 wherein the paeoniflorin preparationcomprises a concentration of paeoniflorin of about 0.25 mg/0.5 ml. 5.The method of claim 1 wherein the paeoniflorin preparation comprises aconcentration of paeoniflorin of about 0.2 mg/ml to about 0.3 mg/ml. 6.The method of claim 1 wherein the paeoniflorin preparation comprises aconcentration of paeoniflorin of about 0.5 mg/ml.
 7. An adipolysisenhancing and subcutaneous fat reducing preparation comprising anadipolysis enhancing amount of paeoniflorin in a suitable carrier,wherein the adipolysis enhancing amount of paeoniflorin in thepreparation is a concentration of paeoniflorin of about 0.02 mg/0.5 mlto about 0.25 mg/0.5 ml.
 8. The adipolysis enhancing and subcutaneousfat reducing preparation of claim 7 wherein the carrier is water.
 9. Theadipolysis enhancing and subcutaneous fat reducing preparation of claim7 further comprising a permeant.
 10. The adipolysis enhancing andsubcutaneous fat reducing preparation of claim 7 comprising aconcentration of paeoniflorin of about 0.25 mg/0.5 ml.
 11. Theadipolysis enhancing and subcutaneous fat reducing preparation of claim7, said preparation containing a concentration of paeoniflorin thatincreases cyclic AMP levels in adipocytes compared to cyclic AMP levelsin adipocytes in the absence of the concentration of paeoniflorin. 12.The adipolysis enhancing and subcutaneous fat reducing preparation ofclaim 7 comprising a concentration of paeoniflorin of about 0.2 mg/ml toabout 0.3 mg/ml.
 13. The adipolysis enhancing and subcutaneous fatreducing preparation of claim 7 comprising a liquid preparation.
 14. Theadipolysis enhancing and subcutaneous fat reducing preparation of claim7 comprising a concentration of paeoniflorin of about 1 umol/L to about5 umol/L.
 15. An adipolysis enhancing and subcutaneous fat reducingpreparation comprising an adipolysis enhancing amount of paeoniflorin ina suitable carrier, wherein the adipolysis enhancing amount ofpaeoniflorin in the preparation is about 11 moles/Liter.
 16. Theadipolysis enhancing and subcutaneous fat reducing preparation of claim15 comprising a liquid preparation.
 17. The adipolysis enhancing andsubcutaneous fat reducing preparation of claim 16 wherein the suitablecarrier is water.